Gelatin non-woven structures produced by a non-toxic dry solvent spinning process

ABSTRACT

The present application discloses an alternative method for the formation of non-woven with fibers in the 1 to 200 μm range. Using an aqueous solution of gelatin (optionally with &lt;30% of low molecular weight alcohol) the fibers are ejected utilizing pressurized air emitted from nozzle and the non-woven formed directly from the emitted thin fibers. 
     The gelatin non-woven can be cross-linked by heat-treatment or chemical cross-linking, and the non-woven is biocompatible as measured by fibroblast growth in vitro and wound healing on pigs in vivo.

FIELD OF THE INVENTION

The present invention is in the field of production of scaffolds forhomeostasis, tissue repair and tissue engineering.

BACKGROUND

Gelatin is prepared by denaturation of collagen. Collagen is a triplestranded helix protein found in skin, cartilage and bones of humans andanimals where it serves as a structural component in the extracellularmatrices (ECM). The strands found in collagen are in gelatin found asmixtures together with oligomers, breakdowns and other polypeptidesforming small local collagen-like triple-helical areas.

The typical sources of gelatin for industrial applications are pigs,cows and fish recovered from collagen by hydrolysis. There are severalvarieties of gelatin, the composition of which depends on the source ofcollagen and the hydrolytic treatment used. Type A gelatin results froman acid hydrolysis of typically skin from pigs whereas type B gelatinresults from alkaline hydrolysis of cattle hides and bones.

Gelatin has sol gel properties, which are thermo reversible. Above about37° C. gelatin is in the sol state, whereas below about 37° C. gelatinis in the gel state. The quality of gelatins is commonly characterizedby bloom, e.g. according to AOAC standards gelatin bloom test and BS757.

Gelatin is widely applied in pharmaceuticals, foods, medical dressingsand technical applications e.g. photographic paper. Due to poor fiberforming properties of gelatin, there are few reports of fibers andnon-woven made of pure gelatin and primarily made by electro-spinningusing organic solutions (Huang Z M et al. (2004), Polymer 45,5361-5368)(Zhang et al. (2004) J Biomed Mater Res Part B: Appl Biomater72B: 156-165). One recent report describes electrospinning of gelatindissolved solely in water (Li J. et al., Biomacromolecules 2006, 7,2243-2247). Most often the fiber forming properties of gelatin areimproved by addition of another polymer or by grafting or substitutingchemical groups to the gelatin chain. Of other examples coating offibers with gelatin can be mentioned (Lin FH et al. (2000) MaterialsChemistry and Physics 64, 1889-195) (W003087444A).

The production of fibers from protein solutions has typically reliedupon the use of wet or dry spinning processes (Martin et al. Processingand Characterization of Protein Polymers; McGrath, K. and Kaplan, D.,Ed.; Birkhauser: Boston, 1997, pp. 339-370; Hudson, S. M. The Spinningof Silk-like Proteins into Fibers; McGrath, K. and Kaplan, D., Ed.:Birkhauser: Boston, 1997, pp. 313-337).

Wet spinning, more commonly used, involves the extrusion of a proteinsolution through a spinneret into an acid-salt coagulating bath, whichusually contains aqueous ammonium sulfate, acetic acid, isopropanol, oracetone (Nagura et al. (2002) Polymer Journal, Vol 34, No 10, 761-766),(JP2001089929), (Fukae R et al. (2005) Polymer 46, 11193-11194).Alternatively, dry spinning consists of extrusion into an evaporativeatmosphere. Both approaches yield large diameter fibers, which do notmimic the morphological characteristics of native collagen fibers.Furthermore, both strategies rely on biologically toxic solvent systemsthat preclude the fabrication in real time of hybrid protein-cellconstructs.

By the electro-spinning process it is possible to make fibers of puregelatin. E.g. US 2004/0110439 describes durable, load bearing prostheticmaterials of cross-linked elastin, cross-linked elastin mimetic protein,cross-linked collagen and/or cross-linked gelatin. Fibers with adiameter in the 200-3,000 nm range are electrospun and a non-wovencreated.

However the electro-spinning process has low output and relies on theuse of expensive and harmful solvents. In wound healing application lowdensity products of gelatin is often made by freeze drying, which hasthe disadvantage of being a costly process and a batch process.

U.S. Pat. No. 5,017,324 describes the formation of fibers with particlesby using one or more spray guns intermixing powder, particulate orstrand-like material with the fibrous material to form a non-woven pad.

SUMMARY

The present application discloses an alternative method for theformation of non-woven with fibers in the 1 to 200 μm range. Using anaqueous solution of gelatin (optionally with <30% of low molecularweight alcohol) the fibers are ejected utilizing pressurized air emittedfrom nozzle and the non-woven formed directly from the emitted thinfibers.

The gelatin non-woven can be cross-linked by heat-treatment or chemicalcross-linking, and the non-woven is biocompatible as measured byfibroblast growth in vitro and wound healing on pigs in vivo.

DETAILED DISCLOSURE

A major aspect of the present invention relates to a method of producingfibers of a natural protein structure comprising the steps of:

-   (a) ejecting an aqueous solution of the natural protein structure    through a nozzle, wherein the aqueous solution comprising <25% low    molecular weight alcohol; while-   (b) emitting pressurized air from air jet bores to attenuate or    stretch the natural protein structure fiber; while-   (c) collecting the fibers on a collecting device.

The present method allows very thin fibers to be extruded by a methodthat can be run in commercial scale. The method according to theinvention is equally applicable to protein structures that are poorfiber makers as well as naturally fiber forming proteins. Gelatin haslow cohesive strength and has been hard to manufacture fibers of. Thepresent technique has proven applicable to make gelatin fibers even fromwater without the use of organic co-solvents.

Materials applicable to present invention are natural proteinstructures, alone or in combinations, particular preferred are thoseoriginating from the ECM. Examples of such materials are collagen,keratin, fibrin, elastin, laminin, vimentin, vitronectin, reticulin,fibrinogen and derivatives of these and the like found in a native ordenaturated form. The viscosity of an aqueous solution of gelatin in thesol state depends on concentration and temperature. In general viscositydecreases with decreasing concentration and decreasing temperature.

In a preferred embodiment of the invention the concentration of agelatin with bloom strength 300 is from 10% to 60%, but more preferablyfrom 20% to 40%, but even more preferably from 22% to 30%, but mostpreferably from 24% to 26%. In a preferred embodiment of the inventionthe viscosity of 25% aqueous solution of a gelatin with bloom 300 is inthe range of 1000-2000 mPas at a processing temperature of 40° C. Inanother preferred embodiment of the invention the viscosity of a 25%aqueous solution of a gelatin with bloom 300 at processing temperaturesfrom 40° C. to 70° C. is in the range of 300-2000 mPas. Processingtemperature is preferably below boiling temperature of solvent, e.g.100° C. of water. In another preferred embodiment viscosity of aqueoussolution of a gelatin with bloom 300 in a concentration between 25% and35% is in the range of 300-8000 mPas at processing temperatures from 40°C. to 70° C. In general a 35% solution of gelatin with bloom 300 needsto be processed at a higher temperature than a 25% solution of samegelatin if viscosity during processing needs to be the same. Howeverprocessing at higher temperatures will increase the rate by which dryingof the formed fiber is taken place. Furthermore in general fiberdiameter of the resulting non-woven will be larger when concentration ofgelatin is 35% compared to processing a gelatin solution (again withbloom 300) of 25%. Processing of solutions with concentration of gelatinof bloom 300 below 25% is also possible, and in a preferred embodimentconcentration is 24% of gelatin (bloom 300). With lower concentrationsprocessing with the described method is also possible, however becauseviscosity decreases with decreasing concentration the cohesive strengthof the solution decreases, hence fiber formation is more difficult.Furthermore the formed non-woven will be wetter, and hence less stablein structure until it is dried. If the non-woven is too wet uponformation it will once it has been dried appear more brittle.

As seen in one of the examples of this patent addition of an alcoholdecreases the viscosity of a gelatin solution at least a lowtemperatures.

It is expected that quality of gelatin, e.g. bloom strength, willinfluence appropriate processing conditions of the preferred embodiment.Hence when using various qualities of a natural protein, processingconditions such as concentration of spinning solution and processingtemperature need to be adjusted in order to obtain preferred embodimentof the described method.

In a preferred embodiment the nozzle has an orifice between 0.008 inchand 0.050 inch, such as bigger than 0.010 inch, and smaller than 0.040inch, that is smaller than 0.035 inch. Preferably the nozzle has anorifice in the 0.012 inch to 0.030 inch range. 0.030 inch orifice equalsa diameter of 762 μm.

To enhance the fiber forming properties of gelatin, and lower theviscosity some solvent is added. However, too much solvent can make thesolution sticky and hard to process. It is therefore preferred that theaqueous solution comprises <30% low molecular weight alcohol, such as<25%, or even <10% low molecular weight alcohol (i.e. less than 10% ofthe final aqueous solution is the solvent). It is even more preferredthat the aqueous solution comprises <1% low molecular weight alcohol,that is essentially free from lower molecular weight alcohol. It is alsopreferred that the aqueous solution is essentially free from any organicsolvents.

The low molecular weight alcohol is selected from the group consistingof methanol, ethanol, propanol (1-propanol, 2-propanol), and 1-butanol.

The ejection process takes place when the aqueous solution is ejected asa bead or a droplet.

Non-woven fibrous structures are produced by extruding a materialthrough a nozzle, which due to its structure allows air from nozzlesadjacent to the extruding nozzle to enhance the fiber formation bydrawing and swirling the material.

The pressurized air is emitted from air jet bores. This attenuates orstretches the natural protein structure fiber by letting pressurized airbe ejected from the air jet directed downwardly and substantiallytangential to the nozzle (WO94/04282). The air also dries the fibers.Preferably, the pressurized air is blown from a source as close to theorifice as possible, creating a substantially tangentially, downwardlyoriented pressurized air flow.

The process and the apparatus is disclosed in detail in WO94/04282.

When the aqueous solution is ejected from the nozzle a thin fiber isformed. Given the high surface area to volume ratio of these fibers,solvent evaporation occurs relatively quickly even when operating withaqueous solutions at ambient temperature and atmospheric pressure. It isappropriate to adjust temperature of both ejected polymer and air suchthat the formed fibers are dry enough to maintain the formed structure,but not dried too fast. When fibers are not too rapidly dried thegelatin molecules will have time to orient on a molecular level. This isrelated to the inherent gel-sol properties of gelatin. When subsequenttreating the fibers with heat the fibers will cross-link moreeffectively if the gelatin has been allowed to gel.

The present method avoids the need for biologically toxic solventsystems. Thus, the present process allows real-time fabrication ofhybrid protein-cell constructs, and constructs of biologically activeconstituents: discrete ECM regions.

Gelatin is an example of a poor fiber forming material, which by thedescribed process in this patent can be made into a fibrous non-wovenmaterial.

The gelatin fiber is still wet and sticky when it leaves the nozzle. Thefiber formation is therefore enhanced if the collection of a fiber isnot in a small area, but spread over the collection device. This can beobtained if the fiber ejected from the nozzle hits the collecting deviceat an angle as described in example 4 where the fibers are sprayed onthe inside of an almost vertical rotating cylinder that is close toparallel to the nozzle, or if the collecting device is perpendicular tothe nozzle, it has to move at a sufficient speed to spread the fibers.Too slow a speed will result in the fibers sticking together while stillwet and forming a more film-like structure.

In one aspect of the invention particles are suspended in the aqueoussolution prior to ejection. As the diameter of the nozzle is wider thanthe diameter of the formed fibers, the particles can have any diameter,up to the diameter of the nozzle, or the particles can be smaller thanthe diameter of the fibers. Wet, soft, and pliable particles of evenlarger diameter than the nozzle may be ejected. Thus, in one aspect ofthe invention the particles suspended in the aqueous solution have amean diameter wider than the mean diameter of the fibers.

What happens is that the natural protein structure comes out through asomewhat wide nozzle. The width of the nozzles also allows the particleto come through without clogging the nozzle. The thinness of the fibersis obtained through the combination of the air-flow emitted and theconsequent stretching the fiber as well as the spinning process formingthe non-woven.

As illustrated in FIG. 3, the thin fiber will have bulbs of particles,where the particles are coated with the natural protein structures. Itis preferred that the particles are compatible with the natural proteinstructures, such that coating is strong. That is, the strength of thefiber will be lowered if the particles are not compatible with thenatural protein structures. When the term ‘particle’ is used, itincludes materials in the form of flakes, fibers, particles, powder orthe like.

It is preferred that the particles are ExtraCellular Matrix (ECM)particles. ECM is the non-cellular portion of animal or human tissues.The ECM is hence the complex material that surrounds cells. In broadterms there are three major components in ECMs: fibrous elements(particularly collagen, elastin, or reticulin), link proteins (e.g.fibronectin, laminin) and space-filling molecules (usuallyglycosaminoglycans (GAG's)). ECMs are known to attract cells and topromote cellular proliferation by serving as a reservoir of growthfactors and cytokines as well as providing the cells with a scaffold.

The ECM material can be obtained from any mammal. It could be derivedfrom, but not limited to, intestinal tissue, bladders, liver, spleen,stomach, lymph nodes or skin. ECM may be derived from human cadaverskin, porcine urinary bladder submucosa (UBS), porcine urinary bladdermatrix (UBM), porcine small intestinal submucosa (SIS).

The active components can also be included in the fiber by dissolvingactive components in the gelatin solution before spinning. These activecomponents included in the present method can also be, or contain,biological signal molecules e.g. chemo attractants, cytokines andgrowths factors, polysaccharides, peptides and derivatives of these andthe likes. Examples of such materials could be but are not limited toGAG's (chondroitin sulfate, dermatan sulfate, heparan sulfate,hyaluronan, heparin etc.), thrombin, fibrinogen, fibrin, fibronectin,vitronectin, vimentin.

The particles or active components could either consist of one material,cross-linked if necessary, or found in combinations, mixed orcross-linked together.

It is preferred that the method further comprises the step of formingnon-woven from the fibers as they are ejected.

The non-woven sheet can be made directly by deposition on a collectingdevice. The dimension and size of the sheet can be controlled by motionof the collecting device or the extruding device (the nozzle). Endlesssheets, which subsequently can be cut into any size or dimension, canalso be produced by the method. In fact, it is also possible to maketubular structures. By this process it is possible to make non-wovenobjects of both well-known fiber forming materials as well as poor fiberforming materials.

It is also preferred that the method further comprises the step ofcross-linking the non-woven natural protein structure. Various methodsof cross-linking exist like glutaraldehyde or1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC), butin this context it is particularly preferred that the cross-linking isdone by heat treatment or ultraviolet irradiation or both. Ultravioletirradiation can be done as a post treatment or as an in-line continuoustreatment. Hereby is avoided to use chemicals that are not compatiblewith introduction into the body as tissue replacements or withintroduction onto the body as dressings.

When using the method for producing gelatin non-woven, the best effectof heat treatment is obtained when the produced gelatin fibers are notdried too rapidly such that sufficient time for gelation, which is anorientation taking place on the molecular level, is ensured. This mean,in essence, that the flow and processing temperatures are adjusted toallow for sufficient slow drying to allow gelation. Non-wovens ofgelatin produced and treated this way can be beta sterilized with 25 kGyand still be sufficiently cross-linked. This also applies to otherstructures made of gelatin for instance by freeze-drying. If the dryingof fibers is too rapid cross-linking by subsequent heat treatment willstill occur but to a lesser degree. Similarly, cross-linking by heattreatment of freeze dried structures will occur to a lesser degree, ifthe gelatin solution is not allowed to gel before freezing. One aspectof the invention relates to a process of cross-linking a natural proteinstructure comprising the step allowing the natural protein to gel priorto drying follow by the step freeze-drying.

Even better cross-linking by subsequent heat treatment can be obtainedby adding a polycarboxylic acid to the spinning solution. The benefit isthat cross-linking is not taking place in the solution, as will be thecase with for example glutaraldehyde. Thus a fiber, a non-woven or forinstance a freeze-dried structure can be processed with thecross-linking additive present without activating it. Activation forcross-linking can be done as a simple post treatment neither involvinghazardous wet chemistry nor needs for energy consumption due to dryingof the product nor induction of deformations of the already obtainedstructure by for example swelling. Examples of usable polycarboxylicacid are poly acrylic acids, citric acid, and carboxy-methyl-cellulose(CMC) but not excluding others.

In the present context, we understand that a gelatin fiber non-woven iscross-linked if it does not disintegrate or dissolve when submerged intap water heated above the sol transition state temperature. A simplesuggested method is to submerge a uniform integrated gelatin non-wovenof 20×20 mm and thickness of 0.5 to 1.5 mm and a weight of 20-30 mg in38° C. tap water for 2 hours. If it is cross-linked it will be possibleto pull the sample up in one piece. If it is not cross-linked it will bedissolved or it will disintegrate when being pulled up.

Suitable crosslinkers are polycarboxylic acids, either the free acid orsalts thereof. These could be (but are not limited to): Synthetic:poly(acrylic acid), poly(methacrylic acid), poly(methyl-vinylether-co-maleic anhydride) (various grades of Gantrez AN), copolymers ofacrylic acid and vinylic monomers (vinylpyrrolidone, alkyl vinyl ethersalkylacrylates, alkylmethacrylates, styrene, maleic anhydride, maleicacid, fumaric acid, hydroxyalkylacrylates, hydroxyalkylmethacrylates),copolymers of methacrylic acid and vinylic monomers (vinylpyrrolidone,alkyl vinyl ethers, alkylacrylates, alkylmethacrylates, styrene, maleicanhydride, maleic acid, fumaric acid, hydroxyalkylacrylates,hydroxyalkylmethacrylates) and copolymers and blends of the above;Natural (acidic polysaccharides most preferred): Pectin,carboxymethylcellulose, sodium alginate, gum arabic, Hyaluronic acid,dermatan sulfate, heparin sulfate, heparan sulfate, chondroitin sulfateand blends of the above.

Crosslinking can be chemical crossbinding, where chemical groups reactand form covalent bounds. However, the same effect of stabilization willbe obtained when complexes are formed resulting in in-solubility. Suchcomplex formation is also considered crosslinking herein.

The product obtained through this process has several unique properties.

The product preferably has oriented fibers. This is a direct result of apreferred method of the invention, where the fibers are ejected onto aconveyer belt or other moving device. Depending on the speed of themoving device the proportion of fibers in one direction will vary.However, typically the flexibility of a non-woven according to theinvention has higher flexibility in one direction than in the other. Ifthis is not a desired feature, the product preferably has two layerswith differently oriented fibers.

The product preferably is non-toxic. That is the production process onlyutilizes a minimum of generally accepted solvents (lower molecularweight alcohols) that evaporates, or at least substantially evaporates,as the natural protein structure is ejected and exposed to the emittedair.

The intended use is a bandage or dressing for human or animal bodydefects such as wounds or where non-wovens may serve as a usefulimplant. The use can be as scaffold for tissue repair on the exterior orin the interior of human or animal bodies. The use can be as absorbingdevice in wounds. Furthermore the use can be structural support insidethe body of humans or animals.

The fibers according to the invention will eventually dissolve,degenerate, and degrade when in contact with body fluids. Thedegeneration rate depends on the body fluid and the degree ofcross-linking. This enables the fibers according to the invention to beused as delivery agents, causing controlled release. The substance to bereleased is preferably added to the aqueous solution prior to ejection.Examples of such substances are peptides, hormones, and vitamins.

One aspect of the invention relates to a non-woven sandwich structurecomprising one layer of gelatin with biological active particles on awound contacting surface of the structure and a second layer of gelatin.

The diameter of the fibers is a consequence of the production process:the air-pressure, the ejection speed, the viscosity of the solution. Oneaspect of the invention relates to a non-woven with an average fiberdiameter of 0.5 and 300 μm, such as 10 to 30 μm. In one aspect none ofthe fibers have a diameter of less than 0.5 μm and/or more than 300 μm.In another aspect none of the fibers have a diameter of less then 10 μmand/or more than 30 μm. A related aspect relates to a wound dressingcomprising non-woven gelatin fibers with a mean diameter of 10 to 30 μmwith ECM particles.

One aspect of the invention relates to a non-woven with particles,wherein the average fiber is smaller than the mean diameter of theparticles. This is particularly advantageous to combine the flexibilityof narrow fibers with the need to incorporate bigger molecules, hereexemplified with ECM particles. In one embodiment the average fiberdiameter is between 0.5 and 300 μm. In a related embodiment the meanparticle diameter is between 10 and 30 μm.

One aspect of the invention relates to a sterilized, cross-linkednon-woven of natural protein fibers, such as gelatin fibers. Asillustrated in the examples, beta-radiation and/or heat-treatment aresuitable for sterilization. As illustrated in the examplesheat-treatment optionally in the presence of polycarboxylic acid issuitable for cross-linking.

EXAMPLES Example 1

An aqueous solution of type B porcine gelatin with 260 bloom ofpharmaceutical grade from Gelita in the ratio of 20 g gelatin to 30 gwater and 3 g propanol was prepared. The gelatin was allowed to dissolvein the liquids by heating to 50° C. for several hours. The dissolvedgelatin solution was transferred to a can, which fit a small lab-sizebulk melter built especially for this purpose. The size of the can usedwith the bulk melter is approximately from 0.5 liter to 1 liter. Thebulk melter heats only the upper surface of the material in the can,which then becomes a viscous liquid and therefore can be pumped to adispensing unit, mounted hereto. The dispensing unit is a CF-200Controlled Fiberization Gun provided from Nordson Corp, equipped with anozzle with 0.012 inch orifice and 6 air holes (FIG. 4). The temperatureof the bulk melter can be controlled in its different parts. Thetemperature of the gun can be controlled and the temperature and rate ofthe air is controlled.

The temperature of the gelatin was kept at approx. 50° C. and the airwas not heated. The obtained non-woven was rigid and the resultingfibers had a diameter from 100 to 200 μm.

The air-flow is controlled by a valve. A maximum of approximately 20 latmospheric air per min is used.

Example 2

In a setup similar to the one described in example 1 a nozzle with anorifice of 0.030 inch (6 air holes) was used. The obtained non-woven wassimilar in structure to the one described in example 1.

Example 3

In a setup similar to the one described in example 1 the temperature ofthe gelatin was kept at approximately 92° C., and the air was heated toapproximately 92° C. The obtained non-woven was less rigid than inexample 1 and fibers were approximately 100 μm wide. A similar resultwas obtained using a nozzle with an orifice of 0.030 inch.

Example 4

An aqueous solution of porcine gelatin with bloom 300 from Gelita wasprepared similar to example 1. The solution contained 30% gelatin and 5%propanol. A nozzle with an orifice of 0.018 inch was used with theequipment mentioned in example 1. A fibrous non-woven structure could beobtained when the collecting device was held in a parallel position tothe fiber extruding direction. It was found that an easy way to processa non-woven sheet was when a rotating cylinder was used as a collectingdevice (FIG. 1). In this case the non-woven sheet was collected on theinner vertical surface of the rotating cylinder. While the cylinder wasrotating it was furthermore moved in the vertical direction alternatingfrom an upward movement to a downward movement. When a constant rate ofthe movements of the collecting device was maintained and the rate offiber output was kept constant it was possible to create a non-wovensheet, which has a uniform appearance (FIG. 5).

The powerfulness of this process is seen by the fact that inapproximately 5 minutes a non-woven gelatin with an area of app. 1350cm² and an approximate thickness of app. 2 mm is made.

Example 5

In another experiment similar to example 4 a 30% gelatin solution wasmade in pure water (70%) without alcohol. The fibers of the resultingnon-woven had diameters from 3 to 7 μm (FIG. 2).

Example 6

In another experiment similar to example 5 a 35% gelatin solution wasmade. The fibers of the resulting non-woven had diameters from app. 4 toapp. 17 μm with an average of app. 9 μm. The non-woven was cross-linkedby a heat-treatment. The heat-treatment was done over night in a vacuumoven, which upon evacuation of air was heated to 120° C. Thecross-linked fibers swell upon hydration but do not dissolve, which onthe other hand was seen with untreated gelatin non-woven.

To evaluate the cell morphology and 3D growth of fibroblasts on gelatinfibers, biopsies were punched out and seeded with primary humanfibroblasts (passage 3) on the surface with a density of 2.5×10⁴cells/cm² in a small volume of growth medium (10% FCS in DMEM)containing antibiotics (penicillin, streptomycin and Amphotericin B).The scaffolds were incubated at 37° C. at 5% CO₂ before additionalgrowth medium was added. Evaluation of the cells attachment, morphology,growth and population of the scaffold were performed on day 1, 3 and 7by staining the cells with neutral red followed by evaluation using anLeica DMIRE2 inverted microscope fitted with a Evolution MP cooledcolour camera (Media Cybernetics). Digital images were taken using ImagePro Plus 5.1 software (Media Cybernetics).

The fibroblasts were adhering to the fibers as spindle-shaped cellsgrowing on single fibers except in regions where several fibers werecrossing each other. These cells were growing across the fibers. Therewas a continuous increase in cells number from the start of the study atday 1 to day 7.

Example 7

In another experiment with a setup similar to the one described inexample 6 a 24% gelatin solution was used. In the gelatin solutionparticles of porcine urinary bladder matrix (UBM) was mixed in. The drymatter of the UBM particles was 30% of the dry matter of gelatin. Theaverage particle size of the particles was approximately 150 μm. Anozzle with an orifice of 0.030 inch was used. The fibers werecross-linked by a heat-treatment similar to the one described in example6. The resulting non-woven had fibers with diameter from app. 3 μm toapp. 15 μm with an average of app. 7 μm (FIG. 3).

In order to evaluate the cell morphology and 3D growth of fibroblasts ongelatin fibers+/−UBM particles, biopsies were punched out of each typeof the scaffolds and seeded with primary human fibroblasts (passage 3)on the surface with a density of 2.5×10⁴ cells/cm² in a small volume ofgrowth medium (10% FCS in DMEM) containing antibiotics (penicillin,streptomycin and Amphotericin B). The scaffolds were incubated at 37° C.at 5% CO₂ before additional growth medium was added. Evaluation of thecells attachment, morphology, growth and population of the scaffold wereperformed on day 1, 3 and 7 by staining the cells with neutral redfollowed by evaluation using an Leica DMIRE2 inverted microscope fittedwith a Evolution MP cooled colour camera (Media Cybernetics). Digitalimages were taken using Image Pro Plus 5.1 software (Media Cybernetics).

The cell growth showed on both types of gelatin fibers (+/−UBMparticles) and on all days tested adherent cells growing asspindle-shaped cells. The cells were growing around the fibers and inareas where several fibers were crossing each other the cells werestretching across the fibers. At the first days of the study nodifference was seen between having UBM particles in the scaffold or notbut at day 7 it was apparent that the cells were more dispersed in thescaffold containing UBM particles compared to the pure scaffold and alsocontracted this scaffolds more. There were a continuously increase incells number from day 1 and to day 7.

One large SPF pig (crossbred of Durac, Yorkshire and Danish landrace atLab Scantox, Denmark) had circular full-thickness wounds approximately20 mm in diameter. The non-woven with UBM (20 mm disc), tested induplicates, was carefully applied on top of the wound-bed. To obtainoptimal contact to the wound-bed, each material was held in place by a20 mm pre-wetted foam plug and covered by foam dressings. On day 2 thetop-foam dressing was removed and the foam plug was very carefullyremoved, so as not to disturb the healing and to ensure that the samplematerials remain in full contact with the wound bed. The wounds werecovered by a hydrocolloid dressing (Comfeel Plus) and changed on day 3,6, 8, 10, 12 and 15. Following euthanasia, each wound was cut free as ablock separated from skeletal muscle tissue and fixed in 10% neutralbuffered formalin. The fixed samples were paraffin embedded andsectioned in 5 μm slices stained with haematoxylin and eosin (HE) forgeneral structure of tissue, Masson's trichroma for newly formedcollagen and von Willebrand factor for angiogenesis. The evaluation wasperformed by a trained pathologist at Lab Scantox.

Massive amounts of granulation tissue developed was observed consistingmainly of large numbers of thin-walled blood vessels andfibrocytes/fibroblasts (fibrovascular connective tissue). Moderateamounts of newly formed collagen and slight angiogenesis were present inthe wounds. A minimal presence of foreign material likely to be testitem was recorded and minimal numbers of clear vacuoles were observed inthe profound granulation tissue.

In the superficial parts of the wounds a moderate to marked inflammationwas found. In the deeper parts of the wounds a marked inflammation waspresent. Marked numbers of giant cells were seen and minimal to slighthaemorrhage was recorded.

The re-epithelialisation was slight and the thickness of the epitheliumwas marked in some cases with rete-ridge formation.

In conclusion, no significantly difference in the histopathologicalwound healings parameters assessed were detected between the non-wowenand the untreated control wounds. However a tendency towards more giantcells were seen in the treated wounds compared to control wounds,probably reflecting a foreign reaction to the non-woven, a common andnaturally reaction to materials left in wounds.

Example 8

It was tested whether it was possible to cross-link the gelatinnon-woven by a heat-treatment, which do not involve vacuum. A one hourtreatment at 160° C. was performed at either atmospheric pressure or invacuum (created with an oil pump—the pressure of the setup (oven, tubes,device for precipitating volatiles by freezing) was later measured todecrease below 0.01 mBar. This level is reached after approximately 30minutes). Gelatin non-wovens made similar to the description in example5 were conditioned to different moisture contents before cross-linkingtreatment. The moisture contents were measured with Karl Fischertitration. The degree of cross-linking was measured with a protein assayusing a bicinchoninic acid kit for protein determination (Sigma BCA-1),where the percent of not cross-linked gelatin was evaluated against astandard curve. Untreated fibers (no heat no vacuum) have percentages ofnot cross-linked protein of above 100%.

TABLE 1 Percent of not cross-linked protein in gelatin non-wovens withdifferent initial moisture contents heat-treated for 1 hour at 160° C.in vacuum oven or conventional oven. Untreated non-woven has percentagesabove 100. Moisture content Vacuum oven Conventional oven 5% 30 ± 2 47 ±7 8% 28 ± 2 41 ± 2 20% 30 ± 3 40 ± 1

The results shown in Table 1 demonstrate that heat-treatment undervacuum is more effective towards cross-linking than heat-treatment atambient pressure. Furthermore initial moisture content has no influenceon the degree of cross-linking

Example 9

The following different mixtures (% of weight) were tested using a 300bloom porcine gelatin from Gelita:

Sample Gelatin water solvent a) 60.2% 36.2% 3.6% 1-propanol b)   60%  32%   8% ethanol c)   50%   45%   5% 1-propanol d)   50%   40%  10%1-propanol e)   30%   60%  10% 1-propanol f)   40%   52%   8% 1-propanolg)   40%   52%   8% ethanol h)   40%   52%   8% 2-propanol i)   40%  52%   8% 1-butanol j)   37%   48%  15% 1-propanol k)   37%   48%  15%ethanol l) 34.5% 43.5%  22% 1-propanol m)   35% 62.5% 2.5% 1-propanol n)  35%   60%   5% 1-propanol o)   35% 57.5% 7.5% 1-propanol p)   35%  55%  10% 1-propanol

The conclusion from the fiber drawing experiments done with thedifferent mixtures is that an alcohol enhances the fiber formingproperties of gelatin, and lowers the viscosity. 1-propanol works best.On the other hand the gelatin solution becomes stickier by addition ofan alcohol. From a practical point of view the enhanced stickiness isless desirable with the given laboratory equipment, because problemswith stopping of the nozzle occur more often. This means that althoughfiber can be made the process is not running stable over a longer timeperiod (with the given equipment), but maybe only for a few minutes andin some cases less than a minute.

Example 10

Using the same equipment as described in the previous examples a 30%gelatin with 10% 1-propanol was made into a non-woven. The extrudingunit was kept at a temperature of 92° C. and the air was cooled to −4°C. The collecting device was a hollow metal plate cooled to negativetemperatures by placing dry ice in the interior hollow space of themetal plate. The non-woven was subsequently transferred to a desiccatorin order to dry the non-woven. The obtained structure was lessfleece-like and more compact like a film compared to the non-wovendescribed in example 4, 5, and 6.

Example 11

Example of a two-layered non-woven construct: Gelatin with biologicalactive particles can be deposited first on the collecting device as alayer of fibrous non-woven and subsequently a layer of pure gelatin canbe deposited on the collecting device, on top of the first layer. Thebenefit of this construct is that it can be placed in a wound such thatthe layer containing biological active particles is placed closest tothe wound surface and the second layer of gelatin serves as an absorbinglayer.

Example 12

Gelatin non-woven was made using the equipment described in example 4.The following samples were prepared: pure gelatin (K), gelatin withEsacure (E), and gelatin with ascorbic acid and riboflavin (V). Theaqueous solution consisted of 24% dry matter gelatin (porchine bloom 300from Gelita) with 0.8% of dry matter of Esacure (32% active), which isan ultraviolet curing photoinitiator supplied by Lamberti spa. Othernon-wovens were made similarly but with an aqueous solution of 24%gelatin with 0.24% ascorbic acid and 0.24% riboflavin (C, and B₂vitamin). Non-wovens of pure gelatin were made similarly. The sampleswere processed in order to have approximately similar fiber thicknessand non-woven thickness (FIGS. 6, 7 and 8).

The dissolved gelatin solution was transferred to a can (1 Liter), whichfit a small lab-size bulk melter (example 4). The bulk melter heats onlythe upper surface of the material in the can, which then becomes viscousand therefore can be pumped to a dispensing unit, mounted hereto. Thedispensing unit is a CF-200 Controlled Fiberization Gun provided fromNordson Corp, equipped with a nozzle with 0.012 inch orifice and 6 airholes. The temperature of the bulk melter can be controlled in itsdifferent parts. The temperature of the gun can be controlled and thetemperature and rate of the air is controlled. The pump was set at 1.Air pressure was 1.4 to 1.6 bar, and temperature of air was 70° C. (K),75-80° C. (E), 50° C. (V). A fibrous non-woven structure could beobtained when the collecting device was held in a parallel position tothe fiber extruding direction. It was found that an easy way to processa non-woven sheet was when a rotating cylinder was used as a collectingdevice. In this case the non-woven sheet was collected on the innervertical surface of the rotating cylinder. While the cylinder wasrotating it was furthermore moved in the vertical direction alternatingfrom an upward movement to a downward movement. When a constant rate ofthe movements of the collecting device was maintained and the rate offiber output was kept constant it was possible to create a non-wovensheet, which has a uniform appearance.

Samples of the three different non-woven fibers were given ultravioletirradiation (A FusionI600 H-tube lamp was used for the UV treatments.The distance from lamp to samples was approximately 30 cm). Twointensities, 40% and 100%, were applied for either 30 or 180 seconds. Inorder to test the degree of cross-linking the samples were immersed inPBS buffer at ambient conditions. Untreated samples of all threenon-woven (Esacure, B₂C-vit and control) dissolved during few hours.UV-treated gelatin non-woven containing Esacure remained intact withvisible fibers after four days. UV-treated gelatin non-woven containingB₂C-vitamin also cross-linked but to a lesser degree than the samplescontaining Esacure. The UV-treated control fibers did not dissolvedafter 4 day but gelled and thus a cross-linking effect of UV was clearlyseen as well. In this experiment the less harsh UV treatments, i.e. the40% intensity for 30 s or 180 s, seemed to give better cross-linkingthan the other treatment. Thus 100% intensity for 180 s is too harsh.

In a second experiment the following 10 combinations of 3 treatmentswere given to the K, E and V samples.

The three treatments were:

-   Heat: one hour at 160° C. under vacuum-   UV: 180 seconds at 40% intensity of the UV lamp-   Beta: sterilization by beta-irradiation with a dose of 25 kGy

The 10 combinations were:

1 Untreated control 2 Beta 3 Heat 4 UV 5 Heat, Beta 6 UV, Beta 7 Heat,UV (first heat then UV) 8 UV, Heat (first UV then heat) 9 Heat, UV, Beta10 UV, Heat, Beta

The degree of cross-linking of the 30 different samples were examined byBCA protein assay with four replicates of each sample.

TABLE 2 BCA protein assay—a value of 100 means that 100% of the sampleis not cross-linked. Thus a low number is preferable in terms ofx-linking. Control Vitamin Esacure 1 Untreated control 103 ± 7.1 110 ±4.6 101 ± 1.9 2 Beta  95 ± 16   99 ± 5.0  95 ± 10  3 Heat  51 ± 13   18± 2.1  28 ± 1.9 4 UV 101 ± 6.2 107 ± 5.9  98 ± 5.1 5 Heat-Beta  46 ± 3.6 32 ± 1.6  50 ± 4.3 6 UV-Beta 102 ± 3.7 109 ± 2.8 107 ± 6.0 7 Heat-UV 34 ± 0.7  25 ± 0.8  35 ± 1.6 8 UV-Heat  45 ± 2.4  33 ± 1.1  28 ± 1.0 9Heat-UV-Beta  43 ± 1.5  34 ± 0.8  51 ± 2.4 10 UV-Heat-Beta  79 ± 15   4± 2.1  42 ± 1.2

A statistical analysis reveals significance of all factors except UV. Itis interesting to note that despite the evidence of UV induced x-linkingcould be seen upon hydration in PBS-buffer, the BCA protein assay showedno significant effect of UV. However, cross-linking with heat treatmenthad a positive effect, whereas beta irradiation (sterilization) has anegative effect. Best cross-linking on gelatin is seen with Vitamin B₂and C—primarily due to heat treatment. Acceptable degree ofcross-linking after beta irradiation on gelatin with Vitamin 8₂ and C

Example 13

To follow-up on the obtained results mentioned in example 12 newexperiments were done, where essentially new control fibers wereprocessed with low temperatures in order to compare to the processingtemperature of the fibers containing vitamin. In the first place thetemperature was kept as low as possible when processing the fiberscontaining vitamin in order to avoid destruction of the thermo-sensitivevitamins.

Surprisingly, it was found that fibers with pure gelatin (no vitamin)could be cross-linked just as well as the fibers containing vitaminmentioned in example 12. Negative controls were produced of both vitamincontaining fiber and pure fibers. With negative controls is meant fibersproduced with a high temperature and deliberately fast dried by blowingair on the newly produced fibers on the collecting device. Data is shownbelow.

Table: Fibers were produced with either pure gelatin or with gelatincontaining 1% Vitamin C and 1% vitamin B2 (% dry matter of final nonwoven). The gelatin was a aqueous solution containing 24% gelatin(weight percentage). Processing temperature is the temperature of thepolymer solution. Post air drying is continuous blowing of air on theproduced non-woven in order to dry it rapidly. Cross-linking before betairradiation is done after heat-treating the non-wovens at 160° C. for 1hour under vacuum. A BCA assay is used to quantify the percentage ofuncross-linked protein; thus a number of 100 means that the sample isnot cross-linked, whereas a lower number is quantifies degree ofcross-linking. Cross-linking after beta irradiation are data obtained ofthe above mentioned treatment followed by beta irradiation of the samplewith a dose of 25 kGy. 4 replicates of BCA assay was done and the resultis given as average±standard deviation.

Processing Post air Cross-linking before Cross-linking after temperaturedrying beta irradiation beta irradiation Vitamin 40 yes  58 ± 1.3  87 ±2.9 Vitamin 42 yes  64 ± 13  76 ± 3.2 Vitamin 42 yes  79 ± 3.2  91 ± 2.1Vitamin 40 yes  91 ± 4.4 105 ± 2.1 Vitamin 75 no 101 ± 3.3 102 ± 4.9Control 52 no  22 ± 1.1  42 ± 2.9 Control 75 no  23 ± 1.4  48 ± 2.6Control 44 no  22 ± 0.8  35 ± 1.9 Control 42 yes  87 ± 1.1 102 ± 1.4

By keeping a relatively high processing temperature of 75° C. whenproducing fibers containing vitamins (row 5), the cross-linking waspoor. Vitamin fibers dried fast by the “Post air drying” treatment weresignificantly poorer cross-linked compared to similar fibers in example12. On the other hand the table shows that non-woven of control fibers(of pure gelatin) could be cross-linked to a degree comparable with whatwas found on the vitamin fibers in example 12. By rapidly drying thecontrol fibers processed at a low temperature (lowest rows in the table)the cross-linking was significantly decreased.

Example 14

Processing of gelatin fibers containing two different types ofglucosamine glucans (GAG) was done using the same processing equipmentas described in the previous example. Spinning solutions were preparedfrom a 300 bloom porcine gelatin from Gelita. Aqueous solutions ofgelatin was prepared 24% concentration. After dissolving the gelatinGAG's were mixed in. The dry matter content of GAG is 1% relative to thegelatin dry matter content. A spinning solution was prepared usingeither dermatan sulphate or chondroitin sulphate as GAG. The temperatureof the processing air was 80° C., hence the temperature of the leavingspinning solution was 80° C., but the temperature of the rest of thesetup was kept at 60° C. Cross-linked samples were produced by heattreatment for 1 hour at 160° C. under vacuum.

The amount of sulphated GAG content together with the gelatin fibers canbe measured using the dimethylmethylen blue (DMMB) assay by an increasein OD 525 nm. The DMMB colour solution was prepared according toFarndale et al. (Biochimica et Biophysica Acta 883:173-177, 1986).Briefly, 16 mg 1,9 dimethylmethylene blue was dissolved in 1 L of watercontaining 3.04 g glycine, 2.37 g NaCl and 95 ml 0.1 M HCl, pH 3.0.Release of GAG from the cross-linked and non-cross-linked gelatinfibers+/−GAGs was measured by placing 6 mm biopsies of each type induplicates in a 48 well plate. Two hundred μl of DMMB solution werepoured over the scaffolds corresponding to the amount necessary to coverthe scaffolds. Five minutes later 100 μl of the colour solution from thewells were transferred to a 96 well plate and measured at 525 nm. ASynergy™ HT Multi-Detection Microplate Reader from Bio-Tek was used.

The result of the study showed an immediately release of GAG into thesolution from the non cross-linked gelatin fibers containing both typesof GAG whereas the cross-linked gelatin fibers containing the GAGs andthe gelatin fibers without additives were coloured up without colouringthe surrounding solution (FIG. 9). This means that the heatcross-linking not only cross-links the gelatin fibers but also somehowcross-links the GAGs into the structure.

Evaluation of the growth of fibroblasts on the cross-linked gelatinfibers+/−GAGs, biopsies were punched out of each type of the scaffoldsand seeded with primary human fibroblasts (passage 4) on the surfacewith a density of 2.5×10⁴ cells/cm² in a small volume of growth medium(10% FCS in DMEM) containing antibiotics (penicillin, streptomycin andAmphotericin B). The scaffolds were incubated at 37° C. at 5% CO₂ beforeadditional growth medium was added. Evaluation of the cells attachment,morphology, growth and population of the scaffold were performed on day1, 3 and 7 by staining the cells with neutral red followed by evaluationusing an Leica DMIRE2 inverted microscope fitted with a Evolution MPcooled colour camera (Media Cybernetics). Digital images were takenusing Image Pro Plus 5.1 software (Media Cybernetics).

The fibroblasts were growing with spindle-shaped morphology on all threetypes of gelatin fiber scaffolds (pure gelatin fibers and the twogelatin fiber scaffolds containing two different GAGs) and an increasingin cell number was seen from day 1 and until day 7. The most predominantdifference between the different types of scaffolds was that the cellswere contracting them in different ways. The gelatin fibers containingCS increased in size whereas a contraction by the cells was seen in thegelatin fibers containing DS. The pure gelatin fiber scaffold weresomewhere in between. The effect was happening somewhere between day 3and day 7 and indicates that even through the GAGs are cross-linked intothe gelatin fibers the GAGs are nevertheless accessible to the cells.

Example 15

Abstract

The effect of 3 different poly(carboxylic acids) (CMC, poly(acrylicacid), and pectin) and of pH on the dehydro-thermal crosslinking ofgelatin is examined. The presence of polycarboxylic acids give superiorcrosslinking compared to plain gelatin when the pH is ≦7 and when theconcentration of polycarboxylic acid is ˜9%. Polyacrylic acid is abetter crosslinker than either CMC or pectin, and has an effect at thelower concentrations of 1 and 5%.

Materials and Methods:

-   Freeze Dryer-   Gelatin Gelita SG 724-8 300 bloom from pig-   CMC, Hercules Blanose 12M31P lot 80357-   Pectin, Pomosin LM-12CG-Z-   Poly(acrylic acid), BASF Sokalan CP10S, 45% aqueous solution-   pH-meter-   BCA-assay (Sigma BCA-1)-   Dilute HCl (approx 0.5%)—for adjustment of pH-   Dilute NaOH (approx 0.5%)—for adjustment of pH-   Phosphate buffer: 7.2 g NaCl+1.48 g Na2HPO4+0.43 g KH2PO4 diluted to    1 L with water-   G: 80 g gelatin is dissolved in hot water to 4 L (2% w/v)-   PAA: 8.89 g Sokalan 45% is dissolved to 200 mL (2% w/v)-   CMC: 6 g CMC is dissolved in hot water to 300 mL (2% w/v)-   P: 6 g pectin is dissolved in hot water to 300 mL (2% w/v)-   0% poly(carboxylic acid): 75 ml G is adjusted to pH (4, 5, 6, 7, 8,    9), The volume is adjusted to 100 ml, the solution is poured in an    aluminium mold (ø50 mm), placed at 5° C. until the solution has    gelled, and is frozen at −20° C., and then freeze-dried.-   1% poly(carboxylic acid): To 75 ml G is added 0.75 ml of PAA, CMC or    P with stirring, and pH is adjusted to (6, 7, 8, 9). The volume is    adjusted to 100 ml, the solution is poured in an aluminium mold (ø50    mm), placed at 5° C. until the solution has gelled, and is frozen at    −20° C., and then freeze-dried.-   4.8% poly(carboxylic acid): To 75 ml G is added 3.75 ml of PAA, CMC    or P with stirring, and pH is adjusted to (6, 7, 8, 9). The volume    is adjusted to 100 ml, the solution is poured in an aluminium mold    (ø50 mm), placed at 5° C. until the solution has gelled, and is    frozen at −20° C., and then freeze-dried.-   9.1% poly(carboxylic acid): To 75 ml G is added 7.5 ml of PAA, CMC    or P with stirring, and pH is adjusted to (6, 7, 8, 9). The volume    is adjusted to 100 ml, the solution is poured in an aluminium mold    (ø50 mm), placed at 5° C. until the solution has gelled, and is    frozen at −20° C., and then freeze-dried.    Cross-Linking:

The freeze-dried porous structures are placed in a vacuum oven (160°C./<0.1T) for 1 h.

The table below shows the design of the experiment (all the volumes areadjusted to 100 ml with water after adjustment of the pH):

poly- Konc pH COOH mL Gelatin mL PAA mL CMC mL P 1 0 4 75 2 0 5 75 3 0 675 4 0 7 75 5 0 8 75 6 0 9 75 7 1 6 PAA 75 0.75 8 1 7 PAA 75 0.75 9 1 8PAA 75 0.75 10 1 9 PAA 75 0.75 11 1 6 CMC 75 0.75 12 1 7 CMC 75 0.75 131 8 CMC 75 0.75 14 1 9 CMC 75 0.75 15 1 6 P 75 0.75 16 1 7 P 75 0.75 171 8 P 75 0.75 18 1 9 P 75 0.75 19 4.8 6 PAA 75 3.75 20 4.8 7 PAA 75 3.7521 4.8 8 PAA 75 3.75 22 4.8 9 PAA 75 3.75 23 4.8 6 CMC 75 3.75 24 4.8 7CMC 75 3.75 25 4.8 8 CMC 75 3.75 26 4.8 9 CMC 75 3.75 27 4.8 6 P 75 3.7528 4.8 7 P 75 3.75 29 4.8 8 P 75 3.75 30 4.8 9 P 75 3.75 31 9.1 6 PAA 757.5 32 9.1 7 PAA 75 7.5 33 9.1 8 PAA 75 7.5 34 9.1 9 PAA 75 7.5 35 9.1 6CMC 75 7.5 36 9.1 7 CMC 75 7.5 37 9.1 8 CMC 75 7.5 38 9.1 9 CMC 75 7.539 9.1 6 P 75 7.5 40 9.1 7 P 75 7.5 41 9.1 8 P 75 7.5 42 9.1 9 P 75 7.5

BCA-assay: 2-4 mg scaffold is weighed to a 4 mL screw-cap vial. 3 mLbuffer is added, the vial is closed and placed in a water bath at 60° C.for 1 h, and then shaken at room temperature overnight. The samples arefiltered, and soluble gelatin is determined spectrophotometrically witha BCA-assay (Sigma BCA-1).

Results and Discussion:

In all the graphs (FIGS. 10, 11 and 12), the x-axis is pH and the y-axis% soluble gelatin. A lower % soluble gelatin equals a higher degree ofcross-linking. These values are compared to gelatin without additives atdifferent pH (FIG. 13): There seems to be little influence of the pH onthe degree of cross-linking of pure gelatin. For the polycarboxylicacids, the PAA gives the strongest effects and the cross-linking seemsto be best at the lower pH-values (6-7). Only PAA give an enhancedcross-linking at the lower concentrations, while pectin and CMC onlyhave an effect at the high concentration (9.1%).

Conclusion:

The effect of 3 different poly(carboxylic acids) (CMC, poly(acrylicacid) and pectin) and pH on the dehydro-thermal cross-linking of gelatinis examined. The presence of polycarboxylic acids give superiorcross-linking compared to plain gelatin when the pH is ≦7 and when theconcentration of polycarboxylic acid is ˜9%. Polyacrylic acid is abetter crosslinker than either CMC or pectin, and has an effect at thelower concentrations of 1 and 5%.

Example 16

Viscosities at different temperatures were measured on aqueous solutionsof porcine gelatin from Gelita, bloom 300, using a Brookfield DV-II+viscometer. Concentration of gelatin was from 15% to 35% (weightpercentage). Included was also a solution made of 30% gelatin, 65%water, and 5% n-propanol (weight percentages). The following tablesummarizes the obtained results:

TABLE 3 Viscosities in mPas measured with Brookfield viscometer. 40° C.50° C. 60° C. 70° C. 15% gelatin 73 55 42 33 20% gelatin 302 167 126 10125% gelatin 1566 525 397 320 30% gelatin 18956 1572 1164 936 30%gelatin + 3407 n.d. 1221 885 5% n-propanol 35% gelatin >1000000 73783199 2120

The conclusion is that viscosity increases with concentration ofgelatin, but decreases with higher temperature. Addition of alcohollowers the viscosity close to the sol-gel transition state temperature.Weak fiber formation properties are related to low viscosity.Difficulties with processing due to too high viscosity may be overcomeby raising processing temperature.

Example 17

Spinning solution were prepared by dissolving porcine gelatin fromGelita with bloom 300 in water to a concentration of 25% with either9.1% of carboxylmethyl cellulose, CMC, type 12 M31P from Hercules or9.1% polyacrylic acid, PAA, (Sokalan CP 10S from BASF) (weightpercentages).

The dissolved gelatin solution was transferred to a can (1 Liter), whichfit a small lab-size bulk melter (example 4). The bulk melter heats onlythe upper surface of the material in the can, which then becomes viscousand therefore can be pumped to a dispensing unit, mounted hereto. Thedispensing unit is a CF-200 Controlled Fiberization Gun provided fromNordson Corp, equipped with a nozzle with 0.012 inch orifice and 6 airholes. The temperature of the bulk melter can be controlled in itsdifferent parts. The temperature of the gun can be controlled and thetemperature and rate of the air is controlled. The pump was set at 1(CMC) and 2 (PAA). Air pressure was 1.1 (CMC) and 1.7 (PAA), andtemperature of air was 51° C. (PAA), however varied from 61-74° C. whenprocessing the PAA spinning solution. A fibrous non-woven structurecould be obtained by collecting on the outer surface of a rotatingcylinder. The rotation axis was horizontal. By using a X-Y table therotating cylinder was moved from side to side along the horizontalrotation axis. Distance from nozzle to collecting surface was 30 cm. Thediameter of the rotating cylinder was 30 cm. The collecting device wasplaced under the nozzle such that the extruded fibers hit the rotatingcylinder almost tangentially. Rotational direction was same direction asthe extrusion direction (not against). When a constant rate of themovements of the collecting device was maintained and the rate of fiberoutput was kept constant it was possible to create a non-woven sheet,which has a uniform appearance.

In order to cross-link the resulting non-wovens samples were heattreated for 1 hour at 160° C. under vacuum. Sterilization was done bybeta irradiation of 25 kGy. Quantification of degree of cross-linkingwas done by BCA assay as described in previous examples. The followingresults were obtained:

0 kGy 25kGy Gelatine 24% 24.16 ± 1.73 35.62 ± 1.85 Gelatine 25%, 9.1%CMC 19.86 ± 0.84 21.55 ± 0.74 Gelatin 25%, 9.1% PAA 18.51 ± 1.62 16.33 ±2.10

The conclusion is that the added polycarboxylic acids are effectivecross-linkers and that the cross-linking effect resists beta irradiationat the given doses.

Figures

-   FIG. 1: Labscale production of gelatin non-woven using the inside of    a rotating cylinder as the collecting device.-   FIG. 2: Gelatin non-woven made from 30% aqueous gelatin. Light    microscopy 10× magnification. Scale bar is 50 μm.-   FIG. 3: Gelatin non-woven with UBM particles. Scale bar is 100 μm.-   FIG. 4: Picture showing examples of nozzles used to make gelatin    non-woven. The orifice is found on the top of the raised center    part. There are either 6 or 12 air holes in a circle around the    orifice.-   FIG. 5: Photograph of gelatin non-woven showing its macroscopic    appearance.-   FIG. 6: Gelatin control 10× magnification. Scalebar 50 μm-   FIG. 7: Gelatin vitamin 10× magnification. Scalebar 50 μm-   FIG. 8: Gelatin Esacure 10× magnification. Scalebar 50 μm-   FIG. 9: Release of two different types of GAG from cross-linked and    non-cross-linked gelatin fibers. The figure shows release of GAG    from gelatin fibers, on the y-axis, the % of control; on the x-axis    the Gelatin with 1% CS (first bar); Gelatin with 1% CS cross-linked    (second bar); Gelatin with 1% DS (third bar); and Gelatin with 1% DS    cross-linked (forth bar).-   FIG. 10A: The x-axis is pH and the y-axis % soluble gelatin. A lower    % soluble gelatin equals a higher degree of crosslinking. 1% PAA was    used.-   FIG. 10B: The x-axis is pH and the y-axis % soluble gelatin. A lower    % soluble gelatin equals a higher degree of crosslinking. 1% CMC was    used.-   FIG. 10C: The x-axis is pH and the y-axis % soluble gelatin. A lower    % soluble gelatin equals a higher degree of crosslinking. 1% pectin    was used.-   FIG. 11A: The x-axis is pH and the y-axis % soluble gelatin. A lower    % soluble gelatin equals a higher degree of crosslinking. 4.8% PAA    was used.-   FIG. 11B: The x-axis is pH and the y-axis % soluble gelatin. A lower    % soluble gelatin equals a higher degree of crosslinking. 4.8% CMC    was used.-   FIG. 11C: The x-axis is pH and the y-axis % soluble gelatin. A lower    % soluble gelatin equals a higher degree of crosslinking. 4.8%    pectin was used.-   FIG. 12A: the x-axis is pH and the y-axis % soluble gelatin. A lower    % soluble gelatin equals a higher degree of crosslinking. 9.1% PAA    was used.-   FIG. 12B: the x-axis is pH and the y-axis % soluble gelatin. A lower    % soluble gelatin equals a higher degree of crosslinking. 9.1% CMC    was used.-   FIG. 12C: the x-axis is pH and the y-axis % soluble gelatin. A lower    % soluble gelatin equals a higher degree of crosslinking. 9.1%    pectin was used.-   FIG. 13: The x-axis is pH and the y-axis % soluble gelatin, for    gelatin without additives at different pH.

The invention claimed is:
 1. A method of producing gelatin fiberscontaining glycosaminoglycans comprising the steps of: (a) ejecting anaqueous alcoholic solution of gelatin and glycosaminoglycans through anozzle to form gelatin fibers containing glycosaminoglycans (GAGs),wherein the aqueous alcoholic solution includes less than 25% lowmolecular weight alcohol; while (b) emitting pressurized air from airjet bores into the gelatin fibers containing the GAGs exiting the nozzleto attenuate or stretch the gelatin fiber containing the GAGs; while (c)collecting the gelatin fibers containing the GAGs on a collectingdevice.
 2. The method according to claim 1, wherein the viscosity of theaqueous solution of gelatin is between 1000 and 2000 mPas at aprocessing temperature of 40° C.
 3. The method according to claim 1,wherein the nozzle has an orifice between 0.008 inch and 0.050 inch. 4.The method according to claim 1, wherein the aqueous solution comprisesless than 10% low molecular alcohol.
 5. The method according to claim 1,wherein the aqueous solution comprises less than 1% low molecularalcohol.
 6. The method according to claim 1, wherein the low molecularweight alcohol is selected from the group consisting of methanol,ethanol, 1-propanol, 2-propanol, and 1-butanol.
 7. The method accordingto claim 1, wherein the collection takes place in parallel to theejection.
 8. The method according to claim 1, further comprising thestep of forming a non-woven structure from the gelatin fibers containingGAGs as they are collected.
 9. The method according to claim 1, furthercomprising the step of allowing the gelatin fibers containing GAGs togel prior to drying.
 10. The method according to claim 1, wherein theaqueous gelatin solution further comprises a polycarboxylic acid.